Pharmacy 325 - Wishart Research Group

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6 Δεκ 2012 (πριν από 4 χρόνια και 6 μήνες)

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Pharmacy 325

Nuclear Magnetic Resonance

(NMR) Spectroscopy


Dr. David Wishart

Rm. 2123

Ph. 492
-
0383

david.wishart@ualberta.ca

Hours: anytime after 4 pm

Lecture Notes Available At:


http://redpoll.pharmacy.ualberta.ca


http://www.pharmacy.ualberta.ca/pharm325/

Electromagnetic Spectrum

Electromagnetic Spectrum

Cosmic

X
-
ray

Ultraviolet

Visible

Infrared

Microwave

Radio

l
(nm)

Hz

10
21

10
18

10
15

10
12

10
9

10
6

10
-
3

1 200 500 10
6

10
9

10
12


Different Types of NMR


Electron Spin Resonance (ESR)


1
-
10 GHz (frequency) used in analyzing
free radicals (unpaired electrons)


Magnetic Resonance Imaging (MRI)


50
-
300 MHz (frequency) for diagnostic
imaging of soft tissues (water detection)


NMR Spectroscopy (MRS)


300
-
900 MHz (frequency) primarily used
for compound ID and characterization

NMR in Everyday Life

Magnetic Resonance Imaging

NMR Spectroscopy

Explaining NMR

UV/Vis spectroscopy

Sample

Explaining NMR

Principles of NMR


Measures
nuclear

magnetism or changes
in nuclear magnetism in a molecule


NMR spectroscopy measures the
absorption of light (radio waves) due to
changes in nuclear spin orientation


NMR only occurs when a sample is in a
strong magnetic field


Different nuclei absorb at different
energies (frequencies)

Protons (and other
nucleons) Have Spin


Spin up




Spin down

Each Spinning Proton is
Like a “Mini
-
Magnet”


Spin up




Spin down

N

S

N

S

Principles of NMR

h
n

Low Energy



High Energy

N

N

S

S

Which Elements or
Molecules are NMR Active?


Any atom or element with an odd
number of neutrons and/or an odd
number of protons


Any molecule with NMR active atoms


1
H
-

1 proton, no neutrons, AW = 1


13
C
-

6 protons, 7 neutrons, AW =13


15
N
-

7 protons, 8 neutrons, AW = 15


19
F = 9 protons, 10 neutrons, AW = 19

The NMR Equation

n

=
g
䈯B
p


B = magnetic field strength in Tesla
(1 Tesla = 10,000 Gauss = 1000
kitchen magnets)


g

= magnetic ratio (characteristic of
each nucleus, each atom in a
molecule

Bigger Magnets are Better

low frequency




high frequency

Increasing magnetic field strength

Different Isotopes Absorb at
Different Frequencies

low frequency




high frequency

2
H

15
N

13
C

19
F

1
H

30 MHz 50 MHz 125 MHz 480 MHz 500 MHz

Typical
1
H NMR Spectrum

Absorbance

NMR Units of Measurement


Energies

~ 10
-
6

eV


Wavelength

~ 30 cm
-

100 cm


Frequency

~ 10
8

-

10
9

Hz


Parts per million (
d)


-

ㄲ1灰洠⡦潲(
1
H)

n
obs

-

n
ref

ppm is proportional to frequency

n
ref

x 10
6

ppm =

IR vs. NMR

Absorbance

NMR vs. IR


NMR has narrower peaks relative to IR


NMR yields
far

more information than IR


NMR allows you to collect data on solids
& liquids but
NOT

gases


NMR is more quantitative than IR or UV


NMR samples are easier to prepare


NMR is much less sensitive than IR or UV


NMR spectrometers are very expensive

A Modern NMR Instrument

Radio Wave

Transceiver

NMR Magnet

Magnet Legs

NMR Magnet Cross
-
Section

An NMR Probe

NMR Sample & Probe Coil

A Modern NMR Instrument

Radio Wave

Transceiver

FT NMR

FT

Free Induction Decay

NMR spectrum

Fourier Transformation

F(
w
) = f(t)e dt

iw
t

Converts from units of time to units of frequency

1
H NMR Spectra Exhibit...

8.0 7.0 6.0 5.0 4.0 3.0 2.0 1.0 0.0


Chemical Shifts (peaks at different
frequencies or ppm values)


Splitting Patterns (from spin coupling)


Different Peak Intensities (#
1
H)

Chemical Shifts


Key to the utility of NMR in chemistry


Different
1
H in different molecules exhibit
different absorption frequencies


Arise from the electron cloud effects of
nearby atoms or bonds, which act as little
magnets to shift absorption
n

異u潲o摯睮


Mostly affected by electronegativity of
neighbouring atoms or groups

Characteristic Chemical Shifts

Spin
-
Spin Coupling


Many
1
H NMR spectra exhibit peak
splitting (doublets, triplets, quartets)


This splitting arises from adjacent
hydrogens (protons) which cause the
absorption frequencies of the observed
1
H to jump to different levels


These energy jumps are quantized and
the number of levels or splittings = n + 1
where “n” is the number of nearby
1
H’s

Spin
-
Spin Coupling


C
-

Y


C
-

CH

C
-

CH
2

C
-

CH
3

H


|

H


|

H


|

H


|

singlet doublet triplet quartet

X

Z

X

Z

X

Z

X

Z

J

Spin Coupling Intensities

1

1 1

1 2 1

1 3 3 1

1 4 6 4 1

1 5 10 10 5 1

1 1

1

1

2

Pascal’s Triangle

1

1

3 3

NMR Peak Intensities




C
-

C
H

C
-

C
H
2

C
-

C
H
3

Y


|

Y


|

Y


|

X

Z

X

Z

X

Z


AUC = 1


AUC = 2


AUC = 3

Applications


Determination of exact structure of drugs
and drug metabolites
-

MOST POWERFUL
METHOD KNOWN


Detection/quantitation of impurities


Detection of enantiomers (shift reagents)


High throughput drug screening


Analysis/deconvolution of liquid mixtures


Water content measurement

Metabonomics


Analysis of blood, urine and other biofluid
mixtures to quantify and identify
metabolite changes


Allows one to detect drug toxicity and
even localize toxicity (for preclinical trials)
in a non
-
invasive way


Detection, identification and quantitation
of primary and secondary drug
metabolites


Metabonomics

Alanine
0.84
mM
Glutamine
0.60
mM
Arginine
0.70
mM
Glycine
1.75
mM
Asparagine
0.72
mM
Hippuric Acid
5.60
mM
Betaine
0.56
mM
Hydroxybutyrate
1.12
mM
Citrate
1.68
mM
Hydroxyproline
1.26
mM
Creatine
4.80
mM
Isoleucine
1.05
mM
Creatinine
16.80
mM
Phenylalanine
1.40
mM
Dimethylamine
1.80
mM
Serine
0.84
mM
Dimethylglycine
3.50
mM
Trimethylamine-N-Oxide
3.00
mM
Other Applications


Clinical testing (detection of inborn
errors of metabolism, cancer,
diabetes, organic solvent poisoning,
drugs of abuse, etc. etc.)


Cholesterol and lipoprotein testing


Chemical Shift Imaging (MRI + MRS)


Pharmaceutical Biotechnology
(proteins, protein drugs, SAR by NMR)